Nocardia sp. ATCC 53492, or an antibiotic A 42867 producing mutant or variant thereof

ABSTRACT

A novel antibiotic, A 42867, is prepared by cultivating the strain Nocardia sp. ATCC 53492, or an antibiotic A 42867-producing mutant or variant thereof, under submerged aerobic conditions in the presence of assimilable sources of carbon, nitrogen and inorganic salts. The antibiotic and its addition salts are useful in the treatment of infectious diseases and as growth promotant agents.

This is a divisional, of application Ser. No. 078,501, filed July 28,1987, now U.S. Pat No. 4,804,534.

The present invention is directed to a new antibiotic substancedenominated antibiotic A 42867, the addition salts thereof, thepharmaceutical compositions thereof and their use as medicaments,particularly in the treatment of infectious diseases involvingmicroorganisms susceptible to them.

The compounds of the invention are also active as growth promotingagents in animals, such as poultry, swine, ruminants, etc.

Another object of the invention is a process for preparing antibiotic A42867 which includes culturing the new strain Nocardia sp. ATCC 53492 oran antibiotic A 42867-producing variant or mutant thereof.

Norcardia sp. ATCC 53492 has been isolated from a soil sample and wasdeposited on May 23, 1986 with the American Type Culture Collection(ATCC), 12301 Parklawn Drive, Rockville, 20852 Maryland, U.S.A. underthe provisions of the Budapest Treaty. The strain has been accordedaccession number ATCC 53492.

In view of their similar antimicrobial activities, reference toantibiotic A 42867 also includes reference to its pharmaceuticallyacceptable addition salt, and vice versa.

The production of antibiotic A 42867 is achieved by cultivating aNocardia sp. capable of producing it, i.e. Nocardia sp. ATCC 53492 or anantibiotic A 42867-producing variant or mutant thereof, under aerobicconditions in an aqueous nutrient medium containing assimilable sourcesof carbon, nitrogen, and inorganic salts. Many of the nutrient mediausually employed in the fermentation art can be used, however certainmedia are preferred. Preferred carbon sources are glucose, mannose,galactose, starch, corn meal and the like. Preferred nitrogen sourcesare ammonia, nitrates, soybean meal, peptone, meat extract, yeastextract, tryptone, aminoacids, and the like. Among the inorganic saltswhich can be incorporated in the culture media there are the customarysoluble salts capable of yielding sodium, potassium, iron, zinc, cobalt,magnesium, calcium, ammonium, chloride, carbonate, sulfate, phosphate,nitrate and like ions.

Ordinarily, the antibiotic-producing strain is pre-cultured in a shakeflask, then the culture is used to inoculate jar fermentors forproduction of substantial quantities of the antibiotic substances. Themedium used for the pre-culture can be the same as that employed forlarger fermentations, but other media can also be employed. Theantibiotic A 42867 producing-strain can be grown at temperatures between20° and 40° C., preferably between 24° and 35° C.

During fermentation, the antibiotic production can be monitored bytesting broth or mycelial extract samples for antibiotic activity, forinstance, by bioassays or TLC or HPLC procedures.

Sensitive organisms to antibiotic A 42867 such as Bacillus subtilis andS. aureus can be used as test organisms. The bioassay is convenientlyperformed by the agar diffusion method on agar plates. Maximumproduction of antibiotic activity generally occurs between the secondand the fifth day of fermentation.

Antibiotic A 42867 is produced by cultivating the strain Nocardia sp.ATCC 53492, or an antibiotic A 42867 producing mutant or varientthereof, and is mainly found in the culture broths.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1--Ultraviolet absorption spectrum of antibiotic A 428671;

FIG. 2--Infrared absorption spectrum of antibiotic A 42867;

FIG. 3--¹ H-NMR spectrum of antibiotic A 42867.

Morphological properties of Nocardia sp. ATCC 53492

The morphology of this strain cultured on soil agar medium is similar tothat one of actinomycetes showing a development of an abundant aerialmycelium with long hyphae moderately branched. A major morphologicalcharacteristic of this strain is the fragmentation of substrate andaerial mycelium in rodlike elements. Fragmentation of substrate myceliumwas observed on all agar media used for the cultural characteristics buton medium No. 5, medium No. 7, Hickey-Tresner, and egg albumin acomplete fragmentation was noted. Substrate hyphae of Nocardia sp. ATCC53492 were fully developed with branched and fragmented rodlike elementsdepending on the age of the culture. Aerial mycelium was well developedon soil agar and on water agar. The aerial hyphae were long straight onsoil agar forming sometimes knots or nest-like tangles. The morphologyof this strain resembles the one of Nocardia genus.

The morphological characterization of this strain was accomplished onthe same plates employed for studying the cultural properties. Toestablish if fragmentation occured on agar plates, the surface of themedium was taken out with a plastic knife and the specimen was observedon a glass slide under an optical microscope. In liquid culture thisstrain showed an extensive fragmentation into bacillary elements.

Cultural characteristics of Nocardia sp. ATCC 53492

For the examination of the cultural characteristics, Nocardia sp. ATCC53492 was cultivated on various standard media suggested by Shirling andGottlieb (Shirling E. B. and Gottlieb D., 1966--Method forcharacterization of Streptomyces species--Int. J. Syst. Bacteriol, 16,313-340) with the addition of several media recommended by Waksman(Waksman, S. A. 1961--The Actinomycetes--The Williams and Wilkins Co.Baltimore; Vol. 2, 328-334).

Color determination was made when necessary by the method of Maerz andPaul (Maerz A. and M. Rea Paul, 1950--A Dictionary of Color--2nd EditionMcGraw-Hill Book Company Inc. New York).

The ability of the organism to utilize different carbon sources wasinvestigated by the method described by Shirling and Gottlieb.

The cultural and physiological characteristics and the carbon sourcesutilization are reported in Tables I, II, III, IV and V.

The readings in Table I have been taken after two week incubation at 28°C.

                  TABLE I                                                         ______________________________________                                        CULTURAL CHARACTERISTICS OF STRAIN                                            Nocardia sp. ATCC 53492                                                       Culture media    Characteristics                                              ______________________________________                                        Medium No. 2     Abundant growth, surface                                     (yeast extract   wrinkled, color brown                                        malt agar)       15/H/11, soluble pigment                                                      yellow                                                       Medium No. 3     Moderate growth, surface                                     (oatmeal agar)   smooth, color orange                                                          12/L/12, trace aerial                                                         mycelium white                                               Medium No. 4     Abundant growth, surface                                     (inorganic salts-                                                                              wrinkled, color orange-dark                                  starch agar)     13/L/12, trace aerial                                                         mycelium white, trace of                                                      soluble pigment color rose                                   Medium No. 5     Abundant growth, surface                                     (glycerol-asparagine                                                                           wrinkled, color orange-dark                                  agar)            13/L/12, trace aerial                                                         mycelium white                                               Medium No. 6     Moderate growth, surface                                     (peptone-yeast extract                                                                         slightly wrinkled, color                                     iron agar)       apricot 10/F/7                                               Medium No. 7     Abundant growth, surface                                     (tyrosine agar)  wrinkled, color brown                                                         15/H/11, trace aerial                                                         mycelium white                                               Czapek-sucrose agar                                                                            Abundant growth, surface                                                      smooth, color yellow-orange                                                   9/G/8, trace aerial                                                           mycelium                                                     Oatmeal agar     Abundant growth, surface                                                      slightly wrinkled, color                                                      orange 12/F/10, trace                                                         aerial mycelium white,                                                        trace of soluble pigment                                                      rose                                                         Hickey and       Abundant growth, surface                                     Tresner's agar   wrinkled, color golden                                                        9/I/6                                                        Calcium malate agar                                                                            Abundant growth, surface                                                      smooth, color pale yellow                                                     10/C/4, trace aerial                                                          mycelium white                                               Bennett's agar   Abundant growth, surface                                                      wrinkled, color brown                                                         15/H/11                                                      Czapek glucose agar                                                                            Abundant growth, surface                                                      wrinkled, color brown                                                         7/E/12, trace aerial                                                          mycelium white, soluble                                                       pigment dark yellow                                          Glucose asparagine                                                                             Moderate growth, surface                                     agar             slightly crusty, color                                                        yellow 10/L/5                                                Nutrient agar    Abundant growth, surface                                                      smooth, color yellow-                                                         orange 9/G/6/                                                Skim milk agar   Abundant growth, surface                                                      smooth, color peach 9/I/5,                                                    trace aerial mycelium white                                  Egg albumin agar Moderate growth, surface                                                      smooth, colorless, trace                                                      aerial mycelium                                              Sabouraud agar   Abundant growth, surface                                                      wrinkled, color brown                                                         10/L/10                                                      Potato agar      Abundant growth, surface                                                      wrinkled, color yellow                                                        10/L/7, trace aerial                                                          mycelium white soluble                                                        pigment rose                                                 Water agar       Very scant growth, surface                                                    smooth, colorless, good                                                       formation of aerial mycelium                                                  white                                                        Soil agar        Moderate growth, surface                                                      smooth, colorless, abundant                                                   formation of aerial mycelium                                 Dextrose tryptone agar                                                                         Abundant growth, surface                                                      wrinkled, color yellow                                                        10/L/7                                                       ______________________________________                                         Letters and numbers refer to the color determined according to Maerz and      Paul (2)                                                                 

Physiological characteristics of Nocardia sp. ATCC 53492

                  TABLE II                                                        ______________________________________                                        Tests               Results                                                   ______________________________________                                        Starch hydrolysis   positive                                                  Tyrosine reaction   positive                                                  Casein hydrolysis   positive                                                  Solubilization of calcium malate                                                                  negative                                                  Nitrite from nitrate                                                                              negative                                                  Cellulose decomposition                                                                           negative                                                  Production of hydrogen sulfide                                                                    positive with lead                                                            acetate strips                                            peptonization       positive                                                  Litmus milk                                                                   coagulation         negative                                                  ______________________________________                                    

Temperature tolerance

                  TABLE III                                                       ______________________________________                                                   15° C. = -                                                             22° C. = +                                                             28° C. = ++                                                            37° C. = ++                                                            42° C. = +                                                             50° C. = -                                                  ______________________________________                                    

The most suitable temperature for the development of the colonies wasfound to range from about 22° C. to about 42° C.

The optimum temperature is from 28° C. to 37° C.

pH tolerance

                  TABLE IV                                                        ______________________________________                                                    pH 3 = -                                                                      pH 4 = -                                                                      pH 5 = ++                                                                     pH 6 = ++                                                                     pH 7 = ++                                                                     pH 8 = ++                                                                     pH 9 = ++                                                                     pH 10 = -                                                                     pH 11 = -                                                         ______________________________________                                         - = No growth                                                                 + = Moderate growth                                                           ++ = Abundant growth                                                     

For physiological characteristics, pH and temperature toleranceexperiments Hickey and Tresner's agar medium was employed.

Utilization of carbon sources

                  TABLE V                                                         ______________________________________                                        Carbon utilization                                                            Carbon Source   Growth                                                        ______________________________________                                        Lactose         ++                                                            Arabinose       ++                                                            Xylose          ++                                                            Mannose         ++                                                            Fructose        ++                                                            Cellobiose      ++                                                            Galactose       ++                                                            Inositol        ++                                                            Glucose         ++                                                            Raffinose        -                                                            Ribose          ++                                                            Sucrose          -                                                            Salicin          +                                                            Cellulose        -                                                            Rhamnose         -                                                            ______________________________________                                         - = no growth                                                                 + = moderate growth                                                           ++ = abundant growth                                                     

For this test medium No. 8 was employed and the results taken after 10days of incubation at 28° C.-30° C.

Chemotaxonomical studies

The strain was cultured in V-6 medium (beef extract 0.5%, autolyzedyeast 0.5%, peptone 0.5%, hydrolized casein 0.3%; glucose 2%, NaCl0.15%), incubated on a rotary shaker at 200 rpm at 30° C. for 72 hours.The mycelium grown in V-6 medium was harvested by centrifugation (3000rpm×10 minutes), and washed twice with distilled water. The mycelium wasfurther washed with ethanol, followed by drying at room temperatureunder a laminar flow.

The dried mycelium was used as a whole-cell preparation.

Amino acids analysis:

The amino acids analysis carried out as described by Becker et al.,("Rapid differentiation between Nocardia and Streptomyces by paperchromatography of whole cell hydrolysates", Appl. Microbiol. 12, 421-423(1964)) showed the presence of meso-diaminopimelic acids.

Sugar analysis:

Analysis of sugar content carried out according to M. P. Lechevalier,("Identification of aerobic actinomycetes of clinical importance", J.Lab. Clin. Med. 71, 934-944 (1968)), using thin layer chromatographysheets as described by J. L. Staneck and G. D. Roberts, ("Simplifiedapproach to identification of aerobic actinomycetes by thin-layerchromatography", Appl. Microbiol., 28, 226-231 (1974), showed thepresence of arabinose and galactose.

Identity of Nocardia sp. ATCC 53492

The presence of meso-diaminopimelic acids together with diagnosticsugars such as galactose and arabinose indicates that this strain is anactinomycetes with cell wall type IV, according to the classification ofLechevalier M. P., and H. Lechevalier, ("Chemical composition as acriterion in the classification of aerobic actinomycetes", Int. Journ.Syst. Bacterial. 20, 435-443 (1970)).

As with other microorganisms, the characteristics of the A 42867producing strain are subject to variation. For example, artificialvariants and mutants of the strain can be obtained by treatment withvarious known mutagens, such as U.V. rays, X-rays, high frequency waves,radioactive rays, and chemicals such as nitrous acid,N-methyl-N'-nitro-N-nitrosoguanidine, and many others. All natural andartificial variants and mutants which belong to the species of the genusNocardia and produce A 42867 antibiotics, are deemed equivalent tostrain Nocardia sp. ATCC 53492 and are contemplated to be within thescope of this invention.

The recovery of A 42867 antibiotics from the fermentation broths of theproducing microorganism is conducted according to known per setechniques which include extraction with solvents, precipitation byadding non-solvents or by changing the pH of the solution, partitionchromatography, reverse-phase partition chromatography, ion-exchangechromatography, affinity chromatography and the like.

A preferred procedure includes an affinity chromatography on immobilizedD-Alanyl-D-Alanine followed by reverse-phase column chromatography.

Immobilized D-Alanyl-D-Alanine matrices suitable for the presentrecovery process are disclosed in European Patent Application No.83112555. The preferred matrix in the present process isD-Alanyl-D-alanine coupled with a controlled pore cross-linkedpolydextrane.

The filtered fermentation broths are then subjected to an affinitychromatography on immobilized D-Alanyl-D-Alanine, either in column orbatchwise.

The binding of the A 42867 antibiotic substance to the affinity matrixis preferably made at a pH of about 7.0-8.0 and its elution is performedat more basic pH values (preferably between 9.0 and 11.5) by means of anaqueous base. This aqueous base may be ammonia, a volatile amine, analkali or alkali metal hydroxide or a basic buffered solution optionallyin the presence of a polar organic solvent such as a polarwater-miscible solvent as defined below.

After removing the impurities by rinsing the column with aqueous bufferpH 4-8, optionally containing salts, urea and/or water misciblesolvents, antibiotic A 42867 is eluted with the above described elutingmixture. The crude antibiotic substance is then recovered preferably byremoving water completely from the pooled antibiotic-containingfractions by azeotropical distillation with an organic solvent capableof forming minimum azeotropic mixtures with water, followed by additionof a non-solvent to precipitate the desired product.

Representative examples of organic solvents capable of forming minimumazeotropic mixtures with water are n-butanol, benzene, toluene, butylether, carbon tetrachloride, chloroform, cyclohexane,2,5-dimethylfurane, hexane and m-xilene; the preferred solvent beingn-butanol.

Examples of non-solvents are: petroleum ether, lower alkyl ethers, suchas ethyl ether, propyl ether and butyl ether, and lower alkyl ketonessuch as acetone.

Alternatively, the pooled antibiotic-containing fractions areconcentrated to a small volume, preferably by azeotropical distillationwith an organic solvent defined as above, and the resulting aqueoussolution is lyophilized.

If the aqueous base employed in the elution is nonvolatile, it may benecessary to neutralize and desalt the concentrate before precipitationor freeze-drying.

A convenient desalting procedure includes applying the antibioticcontaining aqueous solution to a silanized silica gel column, washingwith distilled water and eluting with a mixture of a polarwater-miscible solvent and water.

Representative examples of polar water-miscible solvents are:water-soluble alcohols, (such as methanol, ethanol, iso-propanol,n-butanol), acetone, acetonitrile, lower alkyl alkanoates (such as ethylacetate), tetrahydrofuran, dioxane and dimethylformamide and mixturesthereof; the preferred polar water-miscible solvent being acetonitrile.

Alternatively, desalting may be carried out by applying the antibioticcontaining solution to the above described affinity column, washing withdistilled water and eluting with a volatile aqueous base as describedabove for the elution of the affinity chromatography.

The product so obtained is antibiotic A 42867. A convenient procedure toobtain pure antibiotic A 42867 is represented by a further purificationas obtained above on an affinity chromatography column. The samestationary phase as above (immobilized D-Alanyl-D-Alanine) is generallyused and the desired antibiotic substance is eluted by following theaffinity chromatography procedure on immobilized D-Alanyl-D-Alaninedescribed above.

A preferred immobilized D-Alanyl-D-Alanine isSepharose-ε-aminocaproyl-D-Alanyl-D-Alanine, a preferred equilibratingmixture is 0.16% (w/v) ammonia containing 2M NaCl adjusted to pH 7-8, apreferred rinsing solution is 0.16% (w/v) ammonia containing 2M NaCladjusted to pH 7-8, a preferred eluting mixture is 0.16% (w/v) ammonia.

Alternatively, the antibiotic substance of the invention may be isolatedfrom the fermentation broth or further purified by means of strong orweak anion resins including functionalized polystyrene, acrylic orpolydextrane matrices. Examples of weak anion exchange resins are thosesold under the following trade-names: Dowex MWA-1 or WGR (Dow Chemical),Amberlite IRA-73 (Rohm and Haas), DEAE-Sephadex (Pharmacia). Examples ofstrong anion exchange resins which may be used according to inventioninclude those sold under the following trade names: Dowex MSA-1, SBR,SBR-P (Dow Chemical), Amberlite IR-904 (Rohm and Haas) and QAE-Sephadex(Pharmacia).

The elution of the A 42867 antibiotic substance from these resins isconducted by means of linear gradient mixtures of aqueous solution ofelectrolytes, such as sodium or potassium hydrochlorides, in water ormixtures of water and an organic water-miscible solvent such as a loweralcohol (e. g. (C₁ -C₄)alkanol) or lower alkyl ketones (e.g. acetone,methylethyl ketone, etc.)

Physico-chemical characteristics of antibiotic A 42867

(A) ultraviolet absorption spectrum, which is shown in FIG. 1 of theaccompanying drawings, and exhibits the following absorption maxima:

    ______________________________________                                                             λ max (nm)                                        ______________________________________                                        (a)      0.1 N HCl         282                                                (b)      Water             282                                                (c)      phosphate buffer pH 7.4                                                                         282                                                (d)      phosphate buffer pH 9                                                                           282                                                                           305 (shoulder)                                     (e)      phosphate buffer 0.1 KOH                                                                        305                                                                           265 (shoulder)                                     ______________________________________                                    

(B) infrared absorption spectrum which is shown in FIG. 2 of theaccompanying drawings and exhibits the following absorption maxima(cm⁻¹): 3700-3100, 3000-2800 (nujol); 1650; 1580; 1460 (nujol) 1375(nujol); 1300; 1235; 1210; 1160; 1130; 1060; 1025; 1000; 970; 840;790-700; 720 (nujol)

(C) ¹ H-NMR spectrum which is shown in FIG. 3 and exhibits the followinggroups of signals (in ppm) at 270 MHz recorded in DMSO d₆(hexadeuterodimethylsulfoxide) using TMS as the internal standard (0.00ppm), (δ=ppm): 0.90, d [(CH₃)₂ --(CH)]; 1.02, d [CH₃ --(CH)]; 1.23, d[CH₃ --(CH)]; 1.52, s [CH₃ --C]; 1.77, m [CH(CH₃)₂ ]; 2.38, s (N--CH₃);3.0-6.35, s and m (aromatic, sugar and peptidic CH's); 6.27-9.29(aromatic CH's, peptidic NH's and phenolic OH's)

d=doublet

s=singlet

m=multiplet

(D) retention-time (R_(t)) of 0.537 relative to Vancomycin.HCl(Vancocin, Eli Lilly, R_(t) =16.36 min) when analyzed by reverse phaseHPLC under the following conditions:

column: Ultrasphere ODS (5 μm) Altex (Beckman) 4.6 mm (i.d.)×250 mm

pre-column: Brownlee Labs RP 18 (5 μm)

eluent: Water:acetonitrile:2-ethanolamine:trifluoroacetic acid9:1:0.01:0.01 (v/v)

flow rate: 1.6 ml/min

U.V. detector: 254 nm

internal standard: Vancomycin.HCl (R_(t) =16.36 min) (Vancocin, EliLilly)

(E) retention-time (R_(t)) of 0.665 relative to Vancomycin.HCl(Vancocin, Eli Lilly, R_(t) 9.96 min) when analyzed by reverse phaseHPLC under the following conditions:

column: Ultrasphere ODS (5 μm) Altex (Beckman) 4.6 mm (i.d.)×250 mm

pre-column: Brownlee Labs RP 18 (5 μm)

    ______________________________________                                         eluent A:                                                                             CH.sub.3 CN       2%         adjusted at                                     (2.5 g/l) NaH.sub.2 PO.sub.4.H.sub.2 O                                                         98%         pH 6.0                                    eluent B:                                                                             CH.sub.3 CN      70%         adjusted at                                     (2.5 g/l) NaH.sub.2 PO.sub.4.H.sub.2 O                                                         30%         pH 6.0                                   ______________________________________                                    

elution: linear gradient from 5% to 60% of eluent B in eluent A, in 40min

flow rate: 1.6 ml/min

U.V. detector: 254 nm

internal standard: Vancomycin.HCl (R_(t) =9.96 min) (Vancocin, EliLilly)

(E) elemental analysis, after the sample has been previously dried atabout 140° C. under inert atmosphere which indicates the followingapproximate percentage composition (average): carbon 53.3%; hydrogen5.9%; nitrogen 7.85%; chlorine (total) 4.41%; chlorine (ionic) 2.22%.Inorganic residue at 900° C. in the air: 0.875%.

(G) acid-base titration profile in water upon titration with 0.05Naqueous KOH of a sample previously added with excess of aqueous HClwhich shows pKa values at 3.2, 7.1 and 8.3

(H) R_(f) value of 0.56 in the following chromatographic system:

    ______________________________________                                        (Aqueous sodium chloride)                                                                       70%            adjusted                                     87.5 g/l:NaH.sub.2 PO.sub.4 0.5 g/l                                                                            to pH 6                                      CH.sub.3 CN       30%                                                         ______________________________________                                    

using reverse-phase silanized silica gel plates (RA-18 F₂₅₄)

Visualization:

U V. light at 254 nm

Yellow color with Pauly Reagent, i.e. diazotized sulfanilic acid (J.Chromatog. 20, 171 (1965), Z. Physiol. Chem. 292, 99, (1953))

Bioautography using B. subtilis ATCC 6633 on minimal Davis medium.

(I) MW of about 1559 desumed from a FAB-MS spectrum showing the M+H⊕peak at 1560

Antibiotic A 42867 possesses acid and basic functions and besidesforming internal salts under proper pH conditions can form salts withorganic and inorganic counter-ions according to conventional procedures.

Representative and suitable acid addition salts of the compounds of theinvention include those salts formed by the standard reaction with bothorganic and inorganic acids such as, for example, hydrochloric,hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic,trichloroacetic, succinic, citric, ascorbic, lactic, maleic, fumaric,pamitic, cholic, pamoic, mucic, glutamic, camphoric,glutaric, glycolic,phthalic, tartaric, lauric,stearic, salicylic, methanesulfonic,benzenesulfonic, sorbic, picric, benzoic, cinnamic and the like acids.

Representative examples of these bases are: alkali metal oralkaline-earth metal hydroxide such sodium, potassium, calcium,magnesium, barium hydroxide; ammonia and aliphatic, alicyclic oraromatic organic amines such as methylamine, dimethylamine,trimethylamine, and picoline.

The transformation of the "non-salt" compounds of the invention into thecorresponding addition salts, and the reverse, i.e. the transformationof an addition salt of a compound of the invention into the non-saltform, are within the ordinary technical skill and are encompassed by thepresent invention.

For instance antibiotic A 42867 can be transformed into thecorresponding acid addition-salt by dissolving the non-salt form in anaqueous solvent and adding a slight molar excess of the selected acid orbase. The resulting solution or suspension is then lyophilized torecover the desired salt.

In case the final salt is unsoluble in a solvent where the non-salt formis soluble it is recovered by filtration from the organic solution ofthe non-salt form after addition of the stoichometric amount or a slightmolar excess of the selected acid or base.

The non-salt form can be prepared from a corresponding acid or base saltdissolved in an aqueous solvent which is then neutralized to free thenon-salt form.

When following the neutralization the elimination of the excess of acidor base is necessary, a common desalting procedure may be employed.

For example, column chromatography on silanized silica gel,non-functionalized polystyrene, acrylic and controlled pore polydextraneresins (such as Sephadex LH 20) or activated carbon may be convenientlyused. After eluting the undesired salts with an aqueous solution, thedesired product is eluted by means of a linear gradient or astep-gradient of a mixture of water and a polar or apolar organicsolvent, such as acetonitrile/water from 50:50 to about 100%acetonitrile.

As it is known in the art, the salt formation either withpharmaceutically acceptable acids (or bases) or non-pharmaceuticallyacceptable acids (or bases) may be used as a convenient purificationtechnique. After formation and isolation, the salt form of an A 42867antibiotic can be transformed into the corresponding non-salt form orinto a pharmaceutically acceptable salt form.

In some instances, a base addition salt of antibiotic A 42867 is moresoluble in water and hydrophilic solvents.

The antibacterial activity of the compound of the invention can bedemonstrated in vitro by means of standard dilution tests on differentmicroorganism cultures.

Culture media and growth conditions for MIC (minimal inhibitoryconcentration) determinations were as follows: Isosensitest broth(Oxoid), 24 h, for staphylococci, Strep. faecalis, and Gram-negativebacteria (Escherichia coli); Todd-Hewitt broth (Difco), 24 h for otherstreptococcal species; GC base broth (Difco)+1% Isovitalex (BBL), 48 h,CO₂ -enriched atmosphere for Neisseria gonorrhoeae; Brain Heart broth(Difco)+1% Supplement C (Difco), 48 h for Haemophilus influenzae; ACbroth (Difco), 24 h, anaerobic atmosphere for Clostridium perfringens;Wilkins-Chalgren agar (ref: T. D. Wilkins & S. Chalgren, 1976,Antimicrob. Agents Chemother. 10, 926), 48 h, anaerobic atmosphere forthe other anaerobes (C. difficile, Propionibacterium acnes, Bacteroidesfragilis); PPLO broth (Difco)+10% horse serum+1% glucose, 48 h forMycoplasma gallisepticum; PPLO broth with supplements as in R. T. Evansand D. Taylor-Robinson J. Antimicrob. Chemother. 4, 57, 24 h for U.urealyticum. Incubation was at 37° C. Inocula were as follows: 1% (v/v)of a 48 h broth culture for M. gallisepticum; about 10⁴ color-changingunits/ml for U. urealyticum; about 10⁴ -10⁵ colony-forming units/ml forother broth dilution MICs; about 10⁴ -10⁵ bacteria/spot (inoculated witha multipoint inoculator) for agar dilution MICs (C. difficile, P. acnes,B. fragilis).

The minimal inhibitory concentrations (MIC, μg/ml) for somemicroorganisms are reported below in Table I.

                  TABLE VI                                                        ______________________________________                                                               M.I.C. (μg/ml)                                      Strain                 Antibiotic A 42867                                     ______________________________________                                        Staph. aureus L165 (10.sup.4 cfu/ml)                                                                 0.25                                                   Staph. epidermidis L147 ATCC 12228 (coagulase                                                          1                                                    negative                                                                      Strep. pyogenes L49 C203                                                                             0.13                                                   Strep. pneumoniae L44 UC41                                                                           0.13                                                   Strep. faecalis L149 ATCC 7080                                                                         1                                                    Strep. mitis L796 (clinical isolate)                                                                 0.25                                                   Clostridium perfringens L290 ISS 30543                                                               0.13                                                   Clostridium difficile L1363 ATCC 9689                                                                  2                                                    Propionibacterium acnes L1014 ATCC 6919                                                              0.5                                                    Neisseria gonorrhoeae L997 ISM68/126                                                                 >128                                                   Haemophilus influenzae type b L 970 ATCC                                                             >128                                                   19418                                                                         Proteus vulgaris ATCC 881 L 79                                                                       >128                                                   Escherichia coli L47 SKF 12140                                                                       >128                                                   P. aeruginosa ATCC 10145 L 4                                                                         >128                                                   Candida albicans SKF 2270 L 145                                                                      >128                                                   Mycoplasma gallisepticum L431 S6                                                                     >128                                                   Weybridge                                                                     ______________________________________                                    

Antibiotic A 42867 has been found active against coagulase negativestaphylococci. The M.I.C. (μg/ml) relative to a series of clinicalisolates of S. epidermidis and S. haemolyticus are reported below:

                  TABLE VII                                                       ______________________________________                                                        Antibiotic A 42867                                            Strain          M.I.C. (μg/ml)                                             ______________________________________                                        S. epidermidis L 393                                                                          1                                                             S. epidermidis L 408                                                                          1                                                             S. epidermidis L 410                                                                          0.5                                                           S. haemolyticus L 381                                                                         4                                                             S. haemolyticus L 382                                                                         8                                                             S. haemolyticus L 383                                                                         1                                                             ______________________________________                                    

The antimicrobial activity of the compounds of the invention isconfirmed also in experimental septicemia in the mouse.

Control and treatment groups contained ten CD-1 mice (Charles River)weighing 18-22 g. They are infected intraperitoneally with 0.5 ml ofbacterial suspension prepared by diluting an overnight culture of S.pyogenes C 203 (L 49) with sterile peptonized saline. Inocula wereadjusted so that untreated animals died of septicemia within 48 h. Thecompounds to be tested were administered subcutaneously immediatelyafter infection. On the 7th day, the ED₅₀ in mg/kg was calculated by themethod of Spearman and Karber (D. J. Finney "Statistical Methods inBiological Assay", Griffin, page 524, 1952) from the percentage ofsurviving animals at each dose.

Under these conditions the ED₅₀ value of antibiotic A 42867 was 1.54mg/kg.

In general, for antibacterial treatment antibiotic A 42867 as well asthe non-toxic pharmaceutically acceptable salts thereof or mixturethereof, can be administered by different routes such as topically orparenterally. The parenteral administration is, in general, thepreferred route of administration.

Compositions for injection may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containadjuvants such as suspending, stabilizing and/or dispersing agents.

Alternatively, the active ingredient may be in powder form forreconstitution at the time of delivery when a suitable vehicle, such assterile water, is added thereto.

Depending on the route of administration, these compounds can beformulated into various dosage forms.

In some instances, it may be possible to formulate the compounds of theinvention in enteric-coated dosage forms for oral administration whichmay be prepared as known in the art (see for instance "Remington'sPharmaceutical Sciences", fifteenth edition, Mack Publishing Company,Easton, Pa., U.S.A., page 1614).

This could be especially the case when the absorption of theantimicrobial substance in the enteric tract is particularly desiredwhile passing unaltered through the gastric tract.

The amount of active principle to be administered depends on variousfactors such as the size and condition of the subject to be treated, theroute and frequency of administration, and the causative agent involved.

The antibiotic substances of the present invention and thephysiologically acceptable salts thereof, are generally effective at adaily dosage of between about 0.5 and 50 mg of active ingredient perkilogram of patient body weight, optionally divided into 1 to 4administrations per day.

Particularly desirable compositions are those prepared in dosage unitscontaining from about 100 to about 5,000 mg per unit.

Representative examples of vehicles suitable for injection are: sterilewater for injection, Ringer's solution, 0.9% saline and 5% dextrose. Fori.v. infusion, the suitable concentration of the antibiotic in thevehicle is between about 5% and 10%. Other suitable formulations fordosage units are hermetically sealed vials, plastic pouches, sterile,rubber-stoppered vials and the like.

In addition, the antibiotic substance of the invention can be formulatedin a topical preparation such as a solution, a cream or a lotion. Thesepreparations conveniently contains from 0.1 to 15% (w/v) of the activeingredient.

Furthermore, the antibiotic substances of the invention are useful forsuppressing the growth of Clostridium difficile which causespseudomembranous colitis in the intestine. These antibiotics could beused in the treatment of pseudomembranous colitis by the oraladministration of an effective dose of the antibiotics or apharmaceutically-acceptable salt thereof, prepared in apharmaceutically-acceptable dosage form. For such use, the antibioticscan be administered in gelatin capsules or in liquid suspension.

Besides its activity as medicament, antibiotic A 42867, or an acceptablesalt thereof, can be used as an animal growth promoter.

For this purpose, a compound of the invention is administered orally ina suitable feed. The exact concentration employed is that which isrequired to provide for the active agent in a growth promotant effectiveamount when normal amounts of feed are consumed.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containingthe active ingredient can be blended into the feed.

The way in which such feed premixes and complete rations can be preparedand administered are described in reference books (such as "AppliedAnimal Nutrition", W. H. Freedman and CO., San Francisco, U.S.A., 1969or "Livestock Feeds and Feeding", O and B books, Corvallis, Oreg.,U.S.A., 1977) and are incorporated herein by reference.

The following examples further illustrate the invention and should notbe interpreted as limiting it in any way.

EXAMPLE 1 Production of antibiotic A 42867

The stock culture of the producing organism (Nocardia sp. ATCC 53492) isstreaked on oatmeal agar slants and incubated at 28° C. for 2 weeks. Oneloopful of strain growth is inoculated into a 500 ml Erlenmayer flaskcontaining 100 ml of a seed medium composed of dextrose 2.0%, soybeanmeal 0.8%, yeast extract 0.2%, NaCl 0.1% and CaCO₃ 0.4% whose pH of themedium has been adjusted to 7.3 before sterilization. The flask isincubated on a rotary shaker at 28° C. for 72 hours. A 100 ml aliquot ofthe culture is then inoculated into a jar-fermentor containing 4 litersof the same seed medium and the culture is incubated at 28° C. for 48hours with agitation of about 900 rpm and aeration of one standard literof air per volume per minute. After inoculation of 4 liters of the seedculture into a jar fermentor containing 200 liter of fermentation mediumhaving the same composition as the seed medium, fermentation is carriedout for 96 hours with agitation of about 250 rpm and aeration of onestandard liter of air per volume per minute.

The antibiotic activity was monitored by microbiological assay using B.subtilis cultured on minimal Davis medium.

EXAMPLE 2 Recovery of antibiotic A 42867

The whole fermentation broth (400 liters) obtained as described inExample 1 is filtered using a filter aid (Hyflo-FloMa®), on a rotatoryfilter. The filtered broth is adjusted to pH 7.5 with 2 N hydrochloricacid, and added to 1000 ml of pre-swollenD-Ala-D-Ala-amino-caproyl-Sepharose-4B modified matrix (prepared asdescribed in European Patent Application No. 83112555.4) and leftovernight under slight stirring. The resin is recovered by filtrationand washed with about 10 l of 0.5% (w/v) HCl-Tris buffer pH 7.5 whichcontains 5% (w/v)NaCl and then with water (4×5 l) while the broth isdischarged.

The product selectively bound to the resin is eluted with 1.5% (w/v)ammonia hydroxide (4×5 l) and concentrated to a small volume (about 1800ml) by means of azeotropical distillation with n-butanol under reducedpressure.

The concentrated aqueous solution is lyophilized obtaining crudeantibiotic A 42867 (75.6 g).

EXAMPLE 3 Purification of crude antibiotic A 42867

Crude antibiotic A 42867 obtained by following the procedure of Example2 (75 g) is dissolved in 2 liters of water containing 2 M sodiumchloride, adjusted to pH 7.5 with 0.1 N sodium hydroxide solution, andthen filtered.

The filtrate is applied at 500 ml/hour to a 1000 ml column (0.1×0.1 m)of pre-swollen D-Ala-D-Ala-6-amino-caproyl-Sepharose-4B modified matrix(prepared as described in European Patent Application No. 83112555.4)previously equilibrated with 0.04 M borate buffer pH 7.5 containing 2 Msodium chloride and 0.6 ml of Triton×100 (Baker grade).

The column is washed with 8 l of 8 M urea (pH 7.5) with flow rate of 500ml/h followed by 70 l of aqueous NaOH at pH 10 collecting fractions of1000 ml each. These fractions are assayed on B. subtilis cultures byagar-disc assay and those fractions which are inactive are dischargedwhile those active (like fractions 63-70, in this case) are combined,concentrated to a small volume (500 ml) under reduced pressure by meansof azeotropical distillation with n-butanol and lyophilized to giveantibiotic A 42867 (4 g).

EXAMPLE 4 Purification and desalination of antibiotic A 42867

3.5 g of antibiotic A 42867 obtained by following the procedure ofExample 3 is dissolved in 70 ml of a solution of sodium dihydrogenphosphate monohydrate (2.5 g/l) and acetonitrile (91:9) and filtered. 10ml of this filtrate is applied to a stainless steel column (2×50 cm)packed with 40 g of 10 μm RP 18 Lichrosorb reverse-phase silica gel(Merck). The column is part of a Chromatospac Modulprep unit (JobinYvon, 16-18 Rue de Canal 91169 Longjumeau, France).

The column is eluted at 8 ml/min with the same solution used todissolved the sample and fractions of 50 ml are collected.

Each fraction is monitored by HPLC and paper-disc bioassay onsusceptible microorganisms such as B. subtilis.

The fractions active on B. subtilis of seven runs are combined,acetonitrile is removed by distillation under reduced pressure and theresidue is diluted with a quantity of water which was about the volumeof the initial solution.

The solution is adjusted to pH 7.5 and later applied a flow rate of 100ml/h to a column (5×15 cm) of pre-swollenD-Ala-D-Ala-6-aminocaproyl-Sepharose-4B modified matrix (prepared asdescribed in European Patent Application No. 83112555.4) previouslyequilibrated with 0.04 M borate buffer pH 7.5.

The column is washed with 8 l of water (acidified with 0.5 ml/l of 1 Nhydrochloric acid). The column is then eluted with 1.5% (w/v) ammoniahydroxide collecting fractions of 100 ml each. Those fractions activeagainst B. subtilis are pooled, concentrated under pressure andlyophilized to give 1.2 g of a desalted preparation of antibiotic A42867 whose physico-chemical characteristics ar reported before.

We claim:
 1. A biologically pure culture of Nocardia sp. ATCC 53492 oran antibiotic A 42867 producing mutant or varient thereof, said culturebeing capable of producing antibiotic A 42867 in a recoverable amount inan aqueous nutrient medium containing assimilable sources of carbon,nitrogen and inorganic substances.
 2. The culture of claim 1 which issNocardia sp. ATCC 53492.